Project Summary A20, or TNFAIP3, is a unique dual-function enzyme that is capable of both adding and cleaving ubiquitin chains from target proteins. Ubiquitin chains are essential for intracellular signaling. Specifically, A20?s ubiquitin editing activity is responsible for negative regulation of immune responses and programmed necrosis by restricting NF-kB signaling and RIP signaling, respectively. Inactivating mutations of A20 are associated with lymphoid disorders. Somewhat unexpectedly, however, we have determined that acquired A20 function may be involved in acute myeloid leukemia (AML). AML is characterized by a block in differentiation and acquired self-renewal properties of myeloid progenitor cells, leading to an accumulation of blasts in the bone marrow resulting in anemia, increased susceptibility to infection, and, if untreated, death. While it can occur at any age, it is most common in those over age 60, with prevalence of the diseased expected to increase as the life expectancy of the population increases. Current treatment options include chemotherapy and/or bone marrow transplant, both of which can be taxing even for an otherwise healthy, young individual. A complex interplay of genetic and epigenetic events contributes to the pathogenesis of AML. Despite a better understanding of the mechanisms of AML pathogenesis, new therapy development and identification of therapeutic targets has been limited. From our preliminary findings, A20 is frequently overexpressed in blasts of AML patients and elevated A20 levels in AML patients correlate with a shorter overall survival. Moreover, deletion of A20 in leukemic mouse bone marrow cells impairs leukemic cell function in vitro and prolongs survival in a mouse model of AML. Importantly, the effects of A20 loss were nominal in healthy mouse bone marrow. Furthermore, knockout of A20 in leukemic cells resulted in necrotic signaling and morphology as evidenced by immunoblotting and electron microscopy, respectively, and cell death was rescued by treatment with a necrosis inhibitor. Based on these findings, we predict that A20 contributes to the persistence of leukemic cells through inhibition of an alternative form of cell death, programmed necrosis. Our long-term goal is to understand the mechanisms driving AML development. While A20 has well characterized roles in the immune system, the function of A20 in myeloid leukemia remains unclear. The objectives of this study are (1) to assess A20 function in leukemic stem cells, and (2) to pinpoint the essential ubiquitin-editing function of A20 involved in the pathogenesis of AML. Thus, the proposed project seeks to address a fundamental gap in AML research. In doing so, we will test the following, central hypothesis: Overexpression of A20 in AML assists malignant cells in evading cell death by preventing programmed necrosis. Together, the proposed studies seek to reveal A20?s involvement in the initiation and progression of AML, thereby determining the potential of A20 as a therapeutic target for AML.